[Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references, listed in sequence, may be found at the end of the specification. All of the cited references are incorporated by reference in their entirety.]
Hepatitis B virus (HBV) infection remains a major health problem with more than 350 million chronic HBV carriers worldwide who are at risk for developing liver cirrhosis and hepatocellular carcinoma (HCC) (1, 2, 3, 4). The development of effective therapies for eradicating HBV in chronic carriers has been limited by an incomplete understanding of the mechanisms of viral persistence (5).
HBV is a member of the hepadnavirus family of mammalian hepatitis viruses. HBV is a human virus which can also infect other primates, such as chimpanzee, as well as human hepatocellular carcinoma cells, such as HepG2 and Huh7. Other hepadnaviruses include Woodchuck Hepatitis Virus (WHV) which is native to the woodchuck Marmotta monax and can also infect the ground squirrel, as well as the Ground Squirrel Hepatitis Virus (GSHV) which infects the ground squirrel. Avian hepadnaviruses have been isolated from the duck (DHBV) and the heron (HHBV). In addition to the hepadnaviruses, there are other hepatitis viruses including, for example, Hepatitis A, C, E and F viruses, as well as Hepatitis Delta which requires the presence of Hepatitis B as a helper virus.
Interferon-alpha is the only currently approved treatment for persistent HBV infection (5, 6, 7). Besides exhibiting various immunomodulatory effects (B), interferon-alpha induces the release of intracellular enzymes such as 2′5′-oligoadenylate synthetase and double-stranded RNA-dependent protein kinase, which degrade viral messenger RNAs and inhibit viral protein synthesis (8) in vitro (9) and in vivo (6, 10). Patients who respond to interferon-alpha therapy show a decrease in circulating HBV DNA levels within the first week (8).
Both humoral and cellular elements of the host immune response are important for HBV clearance. The humoral response to HBV antigens, i.e. antibodies to hepatitis B surface antigen (anti-HBs), helps clear circulating virions and confers protection against reinfection, whereas T cell-mediated responses eliminate infected host cells (11, 12). HBV transgenic mice have been developed which replicate wild type HBV under the control of a full length HBV transgene inserted into the mouse genome. Recent work using these HBV transgenic mice has shown that this replication process can be altered by murine cytokines, such as tumor necrosis factor alpha and interferon gamma. These cytokines have the capacity to downregulate HBV replication in a noncytopathic manner (13, 14). It is therefore of interest to determine whether hepatitis virus replication will become persistent in the absence of B and T cells in the host, and whether acute infection of hepatocytes, in such an environment, would lead to viral persistence in all or some cases.
HBV transgenic mice have provided important new information regarding viral pathobiology (11, 12, 13, 14). However, HBV replication in these mice does not occur by an identical mechanism to that which occurs in naturally infected hepatocytes. In the transgenic HBV mice, replication is driven by an integrated transgene in the mouse chromosome. As a result, the hepatocytes can never be completely “cured” of their HBV genomes. In contrast, in hepatocytes which are natural hosts for hepatitis virus infection, replication is normally maintained by a population of episomal covalently closed circular (ccc) viral DNA molecules in the hepatocyte nuclei. These molecules have a limited half life and do not replicate in the nucleus. Therefore, natural host hepatocytes are capable of being completely “cured” of viral DNA. Thus, it would be highly desirable to obtain a system whereby this characteristic of natural host cells could be employed in antiviral testing, since a complete “cure” is possible and could be screened for.
Recently, advances have been made in mouse liver repopulation with transplanted rat hepatocytes (15). In addition, a hepatocyte-lethal phenotype has been discovered in urokinase-type plasminogen activator (uPA) transgenic mice and such mice have been shown to be capable of liver replacement with xenografted rat hepatocytes (16, 17, 18). Such replacement of the mouse liver with xenogenic rat hepatocytes is facilitated in a uPA mouse because uPA transgene expression places these hepatocytes at a growth disadvantage compared with nontransgenic hepatocytes (16). Transplanted hepatocytes in this system are thus selectively amplified in a mixed polyclonal pattern. A disadvantage of this system is that the rat cells are not natural hosts for hepadnaviruses and cannot be infected by natural mechanisms with any of the known hepadnaviruses. It would thus be advantageous to have a method for repopulating the liver parenchyma of many mice with xenogenic mammalian hepatocytes capable of being infected with hepadnaviruses and derived from a single donor, thus creating mice with chimeric livers that contain genetically identical hepatocytes.
The recent isolation of a Severe Combined Immune Deficiency (SCID) mouse which is homozygous for the Recombination Activation Gene 2 (RAG2) knockout mutation provides a mouse deficient in both B and T immune cells (22). This immunetolerant mouse is not capable of rejecting xenogenic tissue.
The woodchuck animal model provides for the study of woodchuck hepatitis virus (WHV) infection in a natural host setting which mimics infection of human liver with HBV (19, 20, 21). One disadvantage of the woodchuck is that it is a relatively inaccessible, genetically heterogeneous animal which is difficult to breed and maintain in a laboratory setting. It would be highly desirable to obtain a model system for hepadna and other hepatitis viral infection in an animal that is both easy to breed and maintain, as well as being genetically controlled and cost effective.
It would be highly desirable to obtain a convenient animal model system for the observation of hepatitis virus replication and development in host hepatocytes that can be infected by natural means. Indeed, it would be highly desirable to use the above model to test the effects of drugs potentially active against hepatitis virus replication, development of hepatocellular carcinoma and treatments for such. A variety of treatments for diseases resulting from hepatitis virus infection could be tested in such an animal model system.